Crystal form i of levornidazole phosphate, and preparation method therefor and use thereof

Abstract

The X-ray powder diffraction pattern of the crystal form I of levornidazole phosphate of the present invention has characteristic peaks at the diffraction angle 2θ of 13.5, 14.7, 17.1, 19.9, 20.1, 20.8, 23.2, 23.4, 23.7, 23.9, 24.2, 25.6, 26.1, 28.0 and 28.3; and the crystal form belongs to the triclinic crystal system and the P1 space group, and the unit cell parameters and the calculated unit cell volume of the crystal form are as follows: (I). The crystal form I of levornidazole phosphate provided in the present invention has a high purity and a good crystal form stability; and a preparation method therefor is simple and convenient, and is suitable for industrial production.

Description

A levo-ornidazole phosphate crystal form I and its preparation method and use Technical Field

[0001] The present invention belongs to the technical field of chemical drug crystallization, and specifically relates to a levornidazole phosphate crystal form I and a preparation method thereof. Background Art

[0002] Ornidazole, a nitroimidazole antibacterial drug, has been widely used against anaerobic and antiprotozoal infections. Following metronidazole, ornidazole is a third-generation nitroimidazole derivative with higher efficacy, shorter treatment duration, improved tolerability, and wider distribution in the body. Ornidazole's antimicrobial activity is achieved through the reduction of the nitro group in its molecular formula to an amino group in an anaerobic environment or through free radical interaction with cellular components, leading to the death of microorganisms.

[0003] Ornidazole is mostly used clinically in the form of large-volume injections. It has extremely poor water solubility and requires strong acidity when made into an infusion preparation. Patients are prone to phlebitis when taking the drug. In addition, the toxic degradation product 2-methyl-5-nitroimidazole produced during high-temperature sterilization of the infusion is relatively high, which brings great safety risks to clinical use.

[0004] To address the poor water solubility of ornidazole products, domestic researchers have made various structural modifications. For example, patent CN200310100057.0 improves solubility by introducing a morpholine ring into the ornidazole structure to form a tertiary amine structure. Patent CN200610166893.2 proposes the structure of left-ornidazole phosphate and its salts, which further improve solubility by phosphorylating the hydroxyl group in the left-ornidazole structure and forming a salt. The process of precipitating left-ornidazole phosphate uses a large amount of organic solvent petroleum ether, which is not conducive to industrial production. Moreover, the stability of left-ornidazole phosphate after salt formation is poor and it is prone to degradation and discoloration.

[0005] Patent CN101177433 mentions the crystalline form and preparation method of levornidazole disodium phosphate pentahydrate, patent CN107151257 mentions levornidazole disodium phosphate hexahydrate and its preparation method, and patent CN109776609 mentions the preparation and use of levornidazole disodium phosphate heptahydrate. The inventors prepared these compounds according to the descriptions in these patents and conducted stability studies under accelerated conditions, and found that these compounds had poor stability.

[0006] As a derivative of ornidazole phosphate, levonidazole disodium phosphate has excellent solubility in water. However, its insufficient stability severely limits its application scenarios. If there is a new, more stable ornidazole phosphate derivative, this deficiency will be well resolved. However, for a long time, due to its extremely strong solubility, the work of obtaining more stable compounds or crystal forms has been stagnant. The only goal that can be achieved is to obtain several compounds containing different crystal water. Although they have a certain degree of stability advantage compared to the original compound, they still cannot solve the problem of the application of this product in the field of solid dosage forms.

[0007] At present, the relevant crystal forms of levonidazole phosphate have not been reported in the literature. It is well known that the crystal form of a drug has an impact on the quality of the preparation and the production process. The study of the crystal form of a drug can provide a reference for formulation workers in terms of prescription development, new dosage form design, optimization of production process, drug quality control and clinical efficacy. Different crystal forms of the same drug may have significant differences in appearance, solubility, melting point, dissolution, bioequivalence, etc., thereby affecting the stability, bioavailability and efficacy of the drug. Therefore, it is necessary to study the crystal form of levonidazole phosphate and develop one or more crystal forms with simple preparation methods, good solubility, high stability, high purity, not easy to absorb moisture, and suitable for industrial production.

[0008] To solve the above problems, the present invention conducts in-depth research on levornidazole phosphate and obtains a levornidazole phosphate crystal form that is soluble in water and has relatively stable physical and chemical properties, providing a basis for safe application.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a levo-ornidazole phosphate crystal form I with a stable spatial structure, thereby providing a new and more stable basis for the clinical application of levo-ornidazole.

[0011] The levoornidazole phosphate crystalline form I provided by the present invention is characterized by having a stable spatial structure, and the specific structural formula is as follows:

[0012] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystalline form I: its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 13.6, 14.7, 16.6, 17.1, 20.0, 20.2, 20.9, 23.0, 23.1, 23.4, 23.7, 23.9, 24.2, 25.7, 26.1, 28.0, 28.4, 29.8, 30.5, 31.3, 31.4, 34.2, 34.8, 37.4, 37.8 and 38.2.

[0013] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystalline form I: its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 13.6, 14.7, 16.6, 17.1, 20.0, 20.2, 20.8, 21.0, 23.0, 23.1, 23.5, 23.7, 24.3, 25.7, 26.1, 27.3, 28.0, 28.4, 29.8, 31.2, 31.5, 34.6, 34.8, 36.1, 37.5 and 38.3.

[0014] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystalline form I: its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 13.6, 14.8, 16.7, 17.1, 20.0, 20.2, 20.9, 23.0, 23.2, 23.4, 23.5, 23.7, 23.9, 24.3, 25.7, 26.1, 27.3, 28.1, 28.4, 29.8, 31.3, 31.6, 34.6, 37.8, 38.3 and 39.5.

[0015] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystalline form I: its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 13.5, 14.7, 16.5, 17.0, 19.9, 20.1, 20.8, 20.9, 22.9, 23.0, 23.2, 23.4, 23.6, 24.2, 25.6, 26.0, 27.2, 27.9, 28.3, 31.3, 31.5, 34.4, 34.6, 36.6, 38.1 and 39.4.

[0016] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystalline form I: its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 8.5, 13.5, 14.7, 16.5, 17.0, 19.9, 20.1, 20.8, 23.0, 23.3, 23.4, 23.6, 23.8, 24.2, 25.7, 26.0, 27.2, 27.9, 28.3, 29.7, 30.4, 31.4, 34.5, 34.7, 36.6 and 38.3.

[0017] The present invention also provides X-ray powder diffraction characteristic peak data of levornidazole phosphate crystal form I: its X-ray powder diffraction spectrum has characteristic peaks at 2θ angles of 13.5, 14.7, 16.6, 17.0, 19.9, 20.1, 20.8, 23.0, 23.3, 23.4, 23.6, 23.8, 24.2, 25.6, 26.0, 28.0, 28.3, 29.7, 31.2, 31.5, 31.8, 34.1, 34.5, 34.7, 36.6 and 37.7

[0018] The present invention provides a levornidazole phosphate crystal form I, and the X-ray powder diffraction pattern preferably has characteristic peaks at 2θ angles of 13.5, 14.7, 17.1, 19.9, 20.1, 20.8, 23.2, 23.4, 23.7, 23.9, 24.2, 25.6, 26.1, 28.0 and 28.3.

[0019] The crystal characteristic peaks in this invention are expressed using methods commonly used in the art. Typically, the tolerance is ±0.2° when accurate to 0.1°. In actual measurements, larger deviations may occur due to particle size, sample height, preferred orientation, or sample transparency, but these deviations should still be understood to fall within the scope of this patent.

[0020] The present application provides a crystalline form I of levornidazole phosphate with unit cell parameters of triclinic system and P1 space group. The unit cell volume calculated by the unit cell parameters is: α=85.249°, β=76.761°, γ=69.451°,

[0021] The single crystal crystallographic structure data and structure refinement parameters of the levornidazole phosphate crystalline form I provided by the present invention are as follows:

[0022] This data is only the test data obtained under the test experimental conditions. The difference in test results caused by changes in the test experimental conditions should also be within the reasonable scope of protection of the present invention.

[0023] The left-ornidazole phosphate crystalline form I of the present invention can be seen from the structural unit schematic diagram and the structural stacking schematic diagram that in its unit cell microstructure, the P=O bonds on two left-ornidazole phosphate molecules interact with P-OH, wherein the P=O bonds of two left-ornidazole phosphates interact with the Cl of the third left-ornidazole phosphate, and the inactive P-OH bonds of two left-ornidazole phosphates interact with the 3-nitrogen on the imidazole ring of the fourth left-ornidazole phosphate.

[0024] The X-ray powder diffraction patterns of levornidazole phosphate crystalline form I are basically shown in Figures 1, 10, 11, 12, 13, and 14.

[0025] The differential scanning calorimetry (DSC) of the levornidazole phosphate crystal form I of the present invention is shown in FIG2 , from which it can be seen that the crystal form has an endothermic peak at a temperature of 180° C. to 210° C.

[0026] The thermogravimetric analysis (TGA) spectrum of the levornidazole phosphate crystal form I of the present invention is shown in FIG3 .

[0027] The X-ray powder diffraction pattern obtained by calculation based on the single crystal diffraction measurement data of levornidazole phosphate form I of the present invention is shown in Figure 4. The calculated X-ray powder diffraction pattern is consistent with the actual measured X-ray powder diffraction pattern.

[0028] The planar morphology of the crystalline form I of levornidazole phosphate of the present invention is shown in FIG5 .

[0029] The schematic diagram of the single crystal structural unit of the levornidazole phosphate crystal form I of the present invention is shown in FIG6 .

[0030] A schematic diagram of the stacking structure of molecules in the single crystal structure of the levornidazole phosphate crystal form I of the present invention (seen from the a-axis direction) is shown in FIG7 .

[0031] A schematic diagram of the stacking structure of molecules in the single crystal structure of the levornidazole phosphate crystal form I of the present invention (seen from the b-axis direction) is shown in FIG8 .

[0032] A schematic diagram of the stacking structure of molecules in the single crystal structure of the levornidazole phosphate crystal form I of the present invention (seen from the c-axis direction) is shown in FIG9 .

[0033] The levornidazole phosphate crystal form I of the present invention has a stable spatial structure. Compared with other crystal forms of the compound, it has better stability, can ensure the quality remains unchanged for a long time, and effectively extends the shelf life of the product.

[0034] The present invention also provides a method for preparing levornidazole phosphate crystal form I, and the specific preparation method is as follows:

[0035] Add a solvent to levornidazole phosphate, heat to dissolve, then add activated carbon, decolorize, filter, and place the filtrate at -5°C to 40°C, stir to precipitate a solid, filter, and place the filter cake at 20°C to 50°C to dry to obtain levornidazole phosphate crystal form I.

[0036] The crystallization temperature in the preparation method is -5℃ to 40℃, which is a preferred condition. It does not mean that crystallization cannot be obtained if it is lower than -5℃ or higher than 40℃. Similarly, the drying temperature of 20℃ to 50℃ is only a preferred condition, which does not mean that the material cannot be dried outside of 20℃ to 50℃.

[0037] The solvent used in the preparation method is one or a mixed solvent of water, methanol, ethanol, isopropanol, n-propanol, and acetone, and the amount of the solvent used is 1 to 100 times the weight of levornidazole phosphate.

[0038] The solvent is a mixed solvent, one of which should be water; the organic solvent is one or more of methanol, acetone, ethanol, n-propanol, and isopropanol.

[0039] The preferred solvent is water, methanol, ethanol aqueous solution, methanol aqueous solution, isopropanol aqueous solution or acetone aqueous solution. The preferred amount of the solvent used is 2 to 20 times the weight ratio of levornidazole phosphate.

[0040] The amount of solvent used in the preparation process is generally 2 to 20 times that of levornidazole phosphate. As professionals in the field of crystallization will understand, similar crystal forms can be obtained by appropriately increasing or decreasing the amount of solvent added, which should also be understood to be within the scope of this patent.

[0041] The levornidazole phosphate (crude product) mentioned in the invention can be obtained by the following method:

[0042] 1. React levornidazole and phosphorus oxychloride in acetonitrile, ethyl acetate or dichloromethane, then add water for hydrolysis, evaporate most of the solvent and water, dissolve the residue in ethanol, add sodium hydroxide solution, adjust the pH to about 2, and stir to precipitate levornidazole phosphate.

[0043] 2. Take levornidazole disodium phosphate and suspend it in ethanol. Add hydrochloric acid to adjust the pH to about 2. Filter to obtain levornidazole phosphate.

[0044] Similarly, methods of interfering with the precipitation process by intentionally adjusting the organic solvent-water ratio, or increasing stirring and shaking during the precipitation process to obtain a product having a certain difference from X-ray powder diffraction should still be understood to be within the scope of this patent.

[0045] In the previous preparation of levornidazole disodium phosphate, the hydrolyzed phosphate was considered difficult to obtain stable crystals, and the ultimate reaction target was levornidazole disodium phosphate. However, the structure of levornidazole disodium phosphate suffers from the disadvantage of poor stability due to the high activity of chlorine.

[0046] In the process of improving the process of levonidazole disodium phosphate, we accidentally noticed that in the process of adjusting the pH of the reactants, as the pH of the solution increased, the solution went through an initial clarification, then gradually became viscous, and then clarified. In previous research and development, this phenomenon was not taken seriously because no effect on the formation of the product was found. In the process of levonidazole disodium phosphate, 4 times the amount of ethanol is usually added to adjust the pH. During the entire desalination and formation of levonidazole phosphate, no levonidazole phosphate derivatives were observed to precipitate. Only after the disodium salt was formed could it be precipitated as a compound containing crystalline water. Since the entire reaction step is carried out in a low-amount of ethanol-water, levonidazole phosphate was previously considered to be a substance with a large solubility in ethanol and water.

[0047] By chance, we treated the prepared 1,2-dimethoate disodium with acid and removed the metal cations. The product was able to precipitate in a suitable solvent. After determining its structure, we confirmed that the substance was 1,2-dimethoate phosphate.

[0048] In subsequent investigations, through the study of the crystal form of levornidazole phosphate, we found that levornidazole phosphate can form a special crystal form through the interaction of P=O and P-OH on the phospholipid groups of different molecules. Compared with levornidazole disodium phosphate, this substance has better stability. Therefore, in practice, levornidazole phosphate can also effectively replace the application scenarios of levornidazole disodium phosphate. More advantageously, it can also fill some application limitations caused by the poor stability of ornidazole disodium phosphate.

[0049] For example, due to its poor stability, levornidazole disodium phosphate is often difficult to use in solid dosage forms such as tablets and capsules. However, the levornidazole phosphate crystalline form exhibits excellent stability and is well suited for the preparation of such formulations. The availability of this crystalline form provides a new, more stable material foundation for the clinical application of ornidazole phosphate derivatives.

[0050] The inventors prepared levornidazole disodium phosphate pentahydrate according to patent CN101177433, prepared levornidazole disodium phosphate hexahydrate according to patent CN107151257, and prepared levornidazole disodium phosphate heptahydrate according to patent CN109776609, and compared their stability with the compounds provided by the present invention. It was found that the stability of the levornidazole phosphate crystal obtained by the present invention was much higher than that of the comparative compound in the form of sodium salt.

[0051] When the levornidazole phosphate crystal form of the present invention is used as an active ingredient in a pharmaceutical composition, it can be prepared into a variety of drug delivery preparations, including various solid drug delivery preparations, due to its good stability.

[0052] The crystal form of the present invention can be used to prevent, improve and treat diseases caused by anaerobic bacteria infection and protozoan infection, and is particularly suitable for use as a human and veterinary medicine.

[0053] The dosage of the levornidazole phosphate of the present invention is 1-100 mg per kg of body weight, preferably 1-20 mg per kg of body weight over a 24-hour period. The optimal treatment regimen is a once-daily administration of 1-50 mg / kg of body weight. To meet the ideal dosing regimen for humans or animals, this dosage can be adjusted upward or downward depending on the severity of the condition and the difficulty of treatment.

[0054] The preparation method of the levornidazole phosphate crystal form I provided by the present invention is simple and suitable for industrial production.

[0055] The levornidazole phosphate crystal form I obtained by the present invention has high purity and good crystal stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG1 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 1 of the present invention;

[0057] FIG2 is a DSC spectrum of Form I of levornidazole phosphate prepared in Example 1 of the present invention;

[0058] FIG3 is a TGA spectrum of levornidazole phosphate crystal form I prepared in Example 1 of the present invention;

[0059] FIG4 is an XRPD pattern calculated from the single crystal structure data of Form I of levornidazole phosphate prepared in Example 1 of the present invention;

[0060] Figure 5 is a planar morphology of the crystalline form I of levonidazole phosphate prepared in Example 1 of the present invention

[0061] FIG6 is a schematic diagram of the single crystal structural unit of Form I of levornidazole phosphate prepared in Example 1 of the present invention;

[0062] FIG7 is a schematic diagram of the stacking structure of molecules in the single crystal structure of levornidazole phosphate Form I prepared in Example 1 of the present invention (seen from the a-axis direction);

[0063] FIG8 is a schematic diagram of the stacking structure of molecules in the single crystal structure of levornidazole phosphate Form I prepared in Example 1 of the present invention (seen from the b-axis direction);

[0064] FIG9 is a schematic diagram of the stacking structure of molecules in the single crystal structure of levornidazole phosphate Form I prepared in Example 1 of the present invention (seen from the c-axis direction);

[0065] Figure 10 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 3 of the present invention;

[0066] FIG11 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 6 of the present invention;

[0067] FIG12 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 9 of the present invention;

[0068] FIG13 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 11 of the present invention;

[0069] FIG14 is an XRPD pattern of Form I of levornidazole phosphate prepared in Example 15 of the present invention;

[0070] Figure 15 is an X-ray diffraction pattern of the levonidazole phosphate powder obtained in the prior art (CN101007823A)

[0071] Figure 16 is an X-ray diffraction pattern of the levonidazole phosphate powder obtained in the prior art (CN1803811A) Specific embodiments

[0072] The following examples will illustrate the implementation of the present invention in more detail, but are not intended to limit the scope of the present invention.

[0073] Example 1: Preparation of Levoornidazole Phosphate Crystalline Form I

[0074] Take 10g of levornidazole phosphate, add 50g of water, heat to 60°C to dissolve, add 0.5g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25°C to crystallize, filter, and dry the solid to 40°C to obtain levornidazole phosphate Form I with a yield of 48% and a purity of 99.9%. The crystal structure, single crystal structure, X-ray powder diffraction pattern, DSC pattern, and TGA pattern are shown in Figures 1-9. The measured unit cell parameters are as follows:

[0075] Example 2: Preparation of Levoornidazole Phosphate Crystalline Form I

[0076] Take 10 g of ornidazole phosphate, add 100 g of water, heat to 60 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 5 ° C for crystallization, filter, and dry the solid to 40 ° C to obtain ornidazole phosphate crystal form I with a yield of 78% and a purity of 100.0%.

[0077] Example 3: Preparation of Levoornidazole Phosphate Crystalline Form I

[0078] Take 10g of levornidazole phosphate, add 50g of mixed solvent (water / ethanol = 1:1, v / v), heat to 70°C to dissolve, add 0.5g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25°C for crystallization, filter, and dry the solid to 40°C to obtain levornidazole phosphate Form I with a yield of 68% and a purity of 99.9%. The X-ray powder diffraction pattern is shown in Figure 10, and the single crystal structure diagram is shown in Figures 6-9. The measured unit cell parameters are as follows:

[0079] Example 4: Preparation of Levoornidazole Phosphate Crystalline Form I

[0080] Take 10 g of ornidazole phosphate, add 100 g of a mixed solvent (water / ethanol = 1:2, v / v), heat to 60 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25 ° C for crystallization, filter, and dry the solid to 40 ° C to obtain ornidazole phosphate Form I with a yield of 72% and a purity of 100.0%.

[0081] Example 5: Preparation of Levoornidazole Phosphate Crystalline Form I

[0082] Take 10 g of left ornidazole phosphate, add 80 g of a mixed solvent (water / ethanol = 1:1, v / v), heat to 60 ° C to dissolve, add 0.3 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 40 ° C to obtain left ornidazole phosphate Form I with a yield of 85% and a purity of 99.9%.

[0083] Example 6: Preparation of Levoornidazole Phosphate Crystalline Form I

[0084] Take 10 g of levornidazole phosphate, add 50 g of a mixed solvent (water / methanol = 1:1, v / v), heat to 70°C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25°C for crystallization, filter, and dry the solid to 50°C to obtain levornidazole phosphate Form I with a yield of 67% and a purity of 99.9%. The X-ray powder diffraction pattern is shown in Figure 11. The measured unit cell parameters are as follows:

[0085] Example 7: Preparation of Levoornidazole Phosphate Crystalline Form I

[0086] Take 10 g of levornidazole phosphate, add 80 g of a mixed solvent (water / methanol = 1:1, v / v), heat to 80 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 40 ° C to obtain levornidazole phosphate Form I with a yield of 90% and a purity of 99.9%.

[0087] Example 8: Preparation of Levoornidazole Phosphate Crystalline Form I

[0088] Take 10 g of left ornidazole phosphate, add 60 g of mixed solvent (water / methanol = 2:3, v / v), heat to 70 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 40 ° C to obtain left ornidazole phosphate Form I with a yield of 88% and a purity of 99.9%.

[0089] Example 9: Preparation of Levoornidazole Phosphate Crystalline Form I

[0090] Take 10 g of levornidazole phosphate, add 50 g of a mixed solvent (water / acetone = 1:1, v / v), heat to 70°C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25°C for crystallization, filter, and dry the solid to 50°C to obtain levornidazole phosphate Form I with a yield of 64% and a purity of 99.9%. The X-ray powder diffraction pattern is shown in Figure 12. The measured unit cell parameters are as follows:

[0091] Example 10: Preparation of Levoornidazole Phosphate Crystalline Form I

[0092] Take 10 g of left ornidazole phosphate, add 100 g of mixed solvent (water / acetone = 1:1, v / v), heat to 60 ° C to dissolve, add 0.4 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 50 ° C to obtain left ornidazole phosphate Form I with a yield of 81% and a purity of 99.9%.

[0093] Example 11: Preparation of Levoornidazole Phosphate Crystalline Form I

[0094] Take 10 g of levornidazole phosphate, add 50 g of a mixed solvent (water / isopropanol = 1:1, v / v), heat to 70°C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25°C for crystallization, filter, and dry the solid to 50°C to obtain levornidazole phosphate Form I with a yield of 63% and a purity of 99.9%. The X-ray powder diffraction pattern is shown in Figure 13. The measured unit cell parameters are as follows:

[0095] Example 12: Preparation of Levoornidazole Phosphate Crystalline Form I

[0096] Take 10 g of ornidazole phosphate, add 80 g of a mixed solvent (water / isopropanol = 1:1, v / v), heat to 70 ° C to dissolve, add 0.4 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 50 ° C to obtain ornidazole phosphate Form I with a yield of 87% and a purity of 99.9%.

[0097] Example 13: Preparation of Levoornidazole Phosphate Crystalline Form I

[0098] Take 10 g of left ornidazole phosphate, add 50 g of a mixed solvent (water / methanol = 2:3, v / v), heat to 70 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 25 ° C for crystallization, filter, and dry the solid to 50 ° C to obtain left ornidazole phosphate Form I with a yield of 66% and a purity of 99.9%.

[0099] Example 14: Preparation of Levoornidazole Phosphate Crystalline Form I

[0100] Take 10 g of ornidazole phosphate, add 50 g of a mixed solvent (water / isopropanol = 1:1, v / v), heat to 70 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 50 ° C to obtain ornidazole phosphate Form I with a yield of 89% and a purity of 99.9%.

[0101] Example 15: Preparation of Levoornidazole Phosphate Crystalline Form I

[0102] Take 10 g of levornidazole phosphate, add 100 g of methanol, heat to 70°C to dissolve, add 1 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0°C for crystallization, filter, and dry the solid to 50°C to obtain levornidazole phosphate Form I with a yield of 81% and a purity of 99.9%. The X-ray powder diffraction pattern is shown in Figure 14, and the single crystal structure diagram is shown in Figures 6-9. The measured unit cell parameters are as follows:

[0103] Example 16: Preparation of Levoornidazole Phosphate Crystalline Form I

[0104] Take 10 g of levornidazole phosphate, add 70 g of a mixed solvent (water / n-propanol = 1:1, v / v), heat to 70 ° C to dissolve, add 0.5 g of activated carbon, stir for 10 minutes, filter, slowly cool the filtrate to 0 ° C for crystallization, filter, and dry the solid to 50 ° C to obtain levornidazole phosphate Form I with a yield of 83% and a purity of 99.9%.

[0105] Comparative Example 1: Preparation of Levo-ornidazole Phosphate according to the method of patent CN101007823A

[0106] Dissolve 110g of L-ornidazole in 500ml of dry ethyl acetate, then add 100ml of phosphorus oxychloride dropwise. Control the reaction temperature between 10 and 20°C. Liquid phase reaction is continued until the peak of L-ornidazole is essentially eliminated. Ethyl acetate is recovered under reduced pressure to obtain a chlorophosphate intermediate. Cool the mixture and slowly add 600ml of purified water. Hydrolysis reaction proceeds for 1 hour. Adjust the pH to 6.0 by slowly adding 10% sodium carbonate solution. Concentrate to dryness under reduced pressure, add 500ml of methanol, filter, and add 500ml of petroleum ether to the filtrate. Freeze to crystallize, and filter to obtain L-ornidazole phosphate. The X-ray powder diffraction pattern is shown in Figure 15.

[0107] Comparative Example 2: Preparation of Levo-ornidazole Phosphate according to the method of patent CN1803811A

[0108] Dissolve 44g of levornidazole in 300ml of acetonitrile, then add 40ml of phosphorus oxychloride dropwise with stirring. Maintain the reaction temperature at 10-15°C. After complete addition, continue the reaction at this temperature for 1 hour. Cool to 0°C for 5 hours to allow crystallization, filter, and dissolve the filter cake in 200ml of water. Hydrolyze for 40 minutes and distill under reduced pressure. Add 300ml of anhydrous ethanol to the residual liquid, freeze-dry for crystallization, and filter to obtain a solid. Dry under vacuum at 30-60°C to obtain ornidazole phosphate. See Figure 16 for the X-ray powder diffraction pattern.

[0109] Comparative Example 3: Preparation of Levo-ornidazole Phosphate Hydrochloride according to the method of patent CN102516298A

[0110] Dissolve 100 g of L-ornidazole phosphate in 500 ml of acetone at 50°C, cool to room temperature, and add concentrated hydrochloric acid dropwise with stirring until almost no solid is produced. Stir for 1 hour and then filter. Dry the solid in a vacuum oven at 40°C to obtain L-ornidazole phosphate hydrochloride.

[0111] Comparative Example 4: Preparation of Levorotatory Ornidazole Disodium Phosphate Pentahydrate

[0112] Levoornidazole disodium phosphate pentahydrate was prepared according to the method provided in patent CN101177433. 300g of Levoornidazole disodium phosphate and 3500ml of 90% ethanol were placed in a reaction flask, heated to 45°C with stirring, and kept stirring for 10 minutes. The mixture was filtered while hot, and the filtrate was cooled to room temperature and then placed at 15°C for 8 hours to allow crystallization. The resulting solid was then filtered, washed with cold ethanol and acetone, and dried at 38°C for 8 hours to obtain Levoornidazole disodium phosphate pentahydrate.

[0113] Comparative Example 5: Preparation of Ornidazole Disodium Phosphate Hexahydrate

[0114] Levoornidazole disodium phosphate hexahydrate was prepared according to the method provided in patent CN107151257. 20g of levonidazole disodium phosphate was dissolved in 120ml of 95% methanol, followed by decolorization with 1.2g of activated carbon. The mixture was stirred for 30 minutes and filtered. The filtrate was incubated at 40°C and 500ml of ethanol was slowly added dropwise with stirring until a solid precipitated. The solid was filtered and dried at 40°C to obtain levonidazole disodium phosphate hexahydrate.

[0115] Comparative Example 6: Preparation of Ornidazole Disodium Phosphate Heptahydrate

[0116] According to the method provided in patent CN109776609, levonidazole disodium phosphate heptahydrate was prepared. 10 g of levonidazole disodium phosphate was added to 500 ml of a mixed solvent (water / ethanol = 1:15, V / V) to dissolve the mixture, equilibrate at 25° C. for half an hour, filter, and filtrate. A small amount of sample was added to the filtrate as seed crystals. The mixture was sealed at 25° C. and then slowly cooled to 10° C., kept at 10° C. for crystallization, and the resulting crystals were collected and dried at room temperature to obtain levonidazole disodium phosphate heptahydrate.

[0117] Experimental Example 1: Comparison of physical and chemical properties

[0118] The physicochemical properties of the levornidazole phosphate crystal form I prepared according to the embodiment of the present invention and the samples prepared in comparative examples 1-3 were compared.

[0119] Solubility determination refers to the classification standards of solubility in the Chinese Pharmacopoeia:

[0120] Very soluble means that 1g (ml) of solute can be dissolved in less than 1ml of solvent;

[0121] Soluble means that 1g (ml) of solute can be dissolved in 1 to less than 10ml of solvent;

[0122] Solubility means that 1g (ml) of solute can be dissolved in 10 to less than 30ml of solvent;

[0123] Slightly soluble means that 1g (ml) of solute can be dissolved in 30 to less than 100ml of solvent;

[0124] Slightly soluble means that 1g (ml) of solute can be dissolved in 100 to less than 1000ml of solvent;

[0125] Very slightly soluble means that 1g (ml) of solute can be dissolved in 1000 to less than 10000ml of solvent;

[0126] Almost insoluble or insoluble means that 1g (ml) of solute cannot be completely dissolved in 10000ml of solvent.

[0127] Test method: Unless otherwise specified, weigh the finely powdered test sample or measure the liquid test sample and shake vigorously for 30 seconds every 5 minutes at 25℃±2℃; observe the dissolution within 30 minutes. If no solute particles or droplets are visible, it is considered to be completely dissolved.

[0128] The criteria for determining hygroscopicity refer to the provisions of the Guiding Principles for Drug Hygroscopicity Test in the Chinese Pharmacopoeia:

[0129] Deliquescent: Absorbs sufficient water to form a liquid.

[0130] Highly hygroscopic: weight gain upon moisture absorption is not less than 15%.

[0131] Hygroscopic: Weight gain due to moisture absorption is less than 15% but not less than 2%.

[0132] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%.

[0133] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0134] Test method:

[0135] 1. Take a dry stoppered glass weighing bottle (outer diameter 50mm, height 15mm) and place it in a suitable 25℃±1℃ constant temperature desiccator (with ammonium chloride or ammonium sulfate saturated solution placed at the bottom) or an artificial climate box (set temperature at 25℃±1℃, relative humidity at 80%±2%) the day before the test, and accurately weigh the weight (m1).

[0136] 2. Take an appropriate amount of the test sample and spread it evenly in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1mm. Accurately weigh the weight (m2).

[0137] 3. Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours.

[0138] 4. Close the weighing bottle lid and accurately weigh the weight (m3).

[0139] Weight gain percentage = (m2-m1) / (m3-m2) x 100%

[0140] The pH value was determined according to the conventional pH determination method, pH meter model: FE28 (Mettler Toledo)

[0141] The results are shown in the table below

[0142] The above results show that the crystalline form I of the present invention has good solubility in water, stable hygroscopicity, and stable solution pH. Although comparative example 1 has good solubility in water, it has poor hygroscopicity and is easily deliquesced, with a solution pH reaching 6.4. According to common sense, the pH of phosphates should not be so high. After studying the preparation process, it was found that sodium carbonate was used to adjust the pH. From this, it was determined that this crystalline form of the phosphate contained some monosodium salt of the phosphate, resulting in a high solution pH. Comparative example 2 also has good solubility in water, but has poor hygroscopicity and is easily deliquesced, with a solution pH of 1.01, while the normal pH of the phosphate should be around 1.7. The reason for this is that a large amount of hydrogen chloride is generated after hydrolysis during the preparation process. This hydrogen chloride reacts with the phosphate to form phosphate hydrochloride, resulting in a low solution pH. Comparative Example 3 has good solubility in water, but poor hygroscopicity and easy deliquesce, with a solution pH of 1.08. This also proves that the levornidazole phosphate hydrochloride prepared in Comparative Example 2 is not levornidazole phosphate. Therefore, the levornidazole phosphate crystal form I of the present invention is superior to the samples of Comparative Examples 1-3.

[0143] Experimental Example 2: Stability Comparison

[0144] Comparison of the stability of the crystal forms prepared in the examples of the present invention and the samples of comparative examples 1-6:

[0145] The stability comparison test of the levornidazole phosphate crystalline form I prepared by the present invention and the samples of comparative examples 1-6 was carried out under the conditions of temperature 60°C ± 2°C and relative humidity 75% ± 5% to examine the relevant substances, contents and residual solvents.

[0146] Chromatographic column: Octadecylsilane bonded silica gel as filler (C18, 250×4.6mm, 5um or equivalent)

[0147] Mobile phase: Mobile phase A, 0.05 mol / L potassium dihydrogen phosphate solution (adjust pH to 3.0 with phosphoric acid), methanol as mobile phase B

[0148] Column temperature: 35°C; flow rate: 1 ml / min; detection wavelength: 321 nm

[0149] The structural formula of impurity A is as follows

[0150] X represents hydrogen or sodium

[0151] The relevant substances, contents and residual solvents were measured on day 0, day 5, day 10 and day 30 respectively. The test results are shown in the table below:

[0152] The above results show that the relevant substances and contents of the levornidazole phosphate crystalline form I of the present invention after storage at 60°C for 30 days are superior to those of the samples of Comparative Examples 1 to 6. The samples prepared in Comparative Examples 2 and 3 have large amounts of residual solvent, which is not conducive to clinical application.

[0153] Experimental Example 3: Comparison of Stability of Levo-ornidazole Phosphate Crystalline Form I

[0154] Take an appropriate amount of levornidazole phosphate crystalline form I prepared according to the method described in Examples 1-16 and place it in a 20 mL colorless transparent glass bottle. Place the sample bottle under the following conditions:

[0155] High temperature: 60℃, high humidity: 92.5%RH, light: 4500lux, acceleration: 40℃75%RH;

[0156] After 2 weeks, the crystals were taken out, their appearance was observed, and XRPD characterization was performed to investigate the physical stability of Form I.

[0157] The stability results of Form I are shown in the table below.

[0158] The above results show that the levornidazole phosphate crystal form I of the present invention has good stability and does not change in crystal form under high temperature, high humidity and light conditions.

[0159] Experimental Example 4: In vivo efficacy study of levornidazole phosphate crystal form I

[0160] Experimental samples: samples prepared in the present invention. Experimental methods: clinical isolates were used to establish a systemic infection model in mice, and the tail vein was used for treatment to observe the therapeutic effect. 50 The test steps are as follows:

[0161] 1. Select clinical isolates of Bacteroides, Pulmonaria, Veillonella, Peptostreptococcus, and Clostridium perfringens.

[0162] 2. Each selected test bacteria was prepared into different concentration series with 5% gastrosin and injected into mice by intraperitoneal injection. The 100% minimum lethal dose (100% MLD) was measured. The 100% MLD of each test bacteria was as follows:

[0163] (1) Bacteroides fragilis about 1.9×10 8 CFU / mouse

[0164] (2) P. volucrata: approximately 4.75×10 7 CFU / mouse

[0165] (3) Veillonella about 1.15×10 8 CFU / mouse

[0166] (4) Peptostreptococci: about 7.5×10 7 CFU / mouse

[0167] (5) Clostridium perfringens about 1.65×10 7 CFU / mouse

[0168] 3. Preparation of test drugs: Use sterile physiological saline to prepare the test drugs into 5 doses at a dose ratio of 1:0.7. The dosages are shown in Tables 1 to 5.

[0169] 4. With 2x MLD 100 Each group of mice was intraperitoneally infected with bacterial concentration, with an infection volume of 0.5 ml / mouse. After infection, the drug was accurately prepared into each dose solution with sterile saline before use. The drug was injected twice via the tail vein, half an hour and 6 hours after infection. The sum of the two doses was the administered dose. The mice were observed for seven days, and the number of surviving mice was recorded. ED was analyzed by Bliss method. 50 calculate.

[0170] Table 1 The samples of the present invention have an effect on the ED of Peptostreptococcus 50 Measurement results

[0171] Table 2 The samples of the present invention have an effect on Bacteroides ED 50 Measurement results

[0172] Table 3: Effect of the samples of the present invention on the ED of P. 50 Measurement results

[0173] Table 4 The samples of the present invention have no effect on the ED of Veillonella 50 Measurement results

[0174] Table 5 The samples of the present invention have an effect on Clostridium perfringens ED 50 Measurement results

[0175] In vivo efficacy studies have shown that levonidazole phosphate crystal form I has a definite therapeutic effect on mouse infections caused by common pathogenic anaerobic bacteria such as Bacteroides, Pulmonaria, Veillonella, Streptococcus and Clostridium perfringens.

[0176] The above-described embodiments represent only a few implementations of the present invention and should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. The scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A crystalline form I of levornidazole phosphate, wherein the levornidazole phosphate has the structure shown below: The X-ray powder diffraction spectrum of the levoornidazole phosphate crystalline form I has characteristic peaks at 13.5, 14.7, 17.1, 19.9, 20.1, 20.8, 23.2, 23.4, 23.7, 23.9, 24.2, 25.6, 26.1, 28.0 and 28.3 at 2θ±0.

2.

2. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levornidazole phosphate crystalline form I more specifically has characteristic peaks at 2θ±0.2 at 13.6, 14.7, 16.6, 17.1, 20.0, 20.2, 20.9, 23.0, 23.1, 23.4, 23.7, 23.9, 24.2, 25.7, 26.1, 28.0, 28.4, 29.8, 30.5, 31.3, 31.4, 34.2, 34.8, 37.4, 37.8 and 38.

2.

3. The levoornidazole phosphate crystalline form I according to claim 2, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is substantially as shown in FIG1 .

4. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levoornidazole phosphate crystalline form I more specifically has characteristic peaks at 2θ±0.2 at 13.6, 14.7, 16.6, 17.1, 20.0, 20.2, 20.8, 21.0, 23.0, 23.1, 23.5, 23.7, 24.3, 25.7, 26.1, 27.3, 28.0, 28.4, 29.8, 31.2, 31.5, 34.6, 34.8, 36.1, 37.5 and 38.

3.

5. The levoornidazole phosphate crystalline form I according to claim 4, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is substantially as shown in FIG10 .

6. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levoornidazole phosphate crystalline form I more specifically has characteristic peaks at 2θ±0.2 at 13.6, 14.8, 16.7, 17.1, 20.0, 20.2, 20.9, 23.0, 23.2, 23.4, 23.5, 23.7, 23.9, 24.3, 25.7, 26.1, 27.3, 28.1, 28.4, 29.8, 31.3, 31.6, 34.6, 37.8, 38.3 and 39.

5.

7. The levoornidazole phosphate crystalline form I according to claim 6, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is substantially as shown in FIG11 .

8. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levornidazole phosphate crystalline form I more specifically has characteristic peaks at 2θ±0.2 at 13.5, 14.7, 16.5, 17.0, 19.9, 20.1, 20.8, 20.9, 22.9, 23.0, 23.2, 23.4, 23.6, 24.2, 25.6, 26.0, 27.2, 27.9, 28.3, 31.3, 31.5, 34.4, 34.6, 36.6, 38.1 and 39.

4.

9. The levoornidazole phosphate crystalline form I according to claim 8, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is basically shown in Figure 12.

10. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levoornidazole phosphate crystalline form I more specifically has characteristic peaks at 8.5, 13.5, 14.7, 16.5, 17.0, 19.9, 20.1, 20.8, 23.0, 23.3, 23.4, 23.6, 23.8, 24.2, 25.7, 26.0, 27.2, 27.9, 28.3, 29.7, 30.4, 31.4, 34.5, 34.7, 36.6 and 38.3 at 2θ±0.

2.

11. The levoornidazole phosphate crystalline form I according to claim 10, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is basically shown in Figure 13.

12. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern of the levoornidazole phosphate crystalline form I more specifically has characteristic peaks at 2θ±0.2 at 13.5, 14.7, 16.6, 17.0, 19.9, 20.1, 20.8, 23.0, 23.3, 23.4, 23.6, 23.8, 24.2, 25.6, 26.0, 28.0, 28.3, 29.7, 31.2, 31.5, 31.8, 34.1, 34.5, 34.7, 36.6 and 37.

7.

13. The levoornidazole phosphate crystalline form I according to claim 12, characterized in that: The X-ray powder diffraction pattern of the levo-ornidazole phosphate crystalline form I is basically shown in Figure 14.

14. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The unit cell parameters are: triclinic system, P1 space group, and the unit cell parameters are used to calculate the unit cell volume: α=85.249°, β=76.761°, γ=69.451°, 15. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: The unit cell structure is characterized by: the P=O bonds on two left-ornidazole phosphate molecules interact with P-OH, wherein the P=O bonds of two left-ornidazole phosphates interact with the Cl of the third left-ornidazole phosphate, and the inactive P-OH bonds of two left-ornidazole phosphates interact with the 3-nitrogen on the imidazole ring of the fourth left-ornidazole phosphate.

16. The levoornidazole phosphate crystalline form I according to claim 1, characterized in that: When measured by differential scanning calorimetry, the levoornidazole phosphate crystalline form I has an endothermic peak at a temperature of 180°C to 210°C.

17. The method for preparing levoornidazole phosphate crystal form I according to claim 1, characterized in that: The preparation method comprises: adding levornidazole phosphate into a solvent, heating to dissolve, adding activated carbon, decolorizing, filtering, placing the filtrate at -10°C to 40°C, stirring to precipitate solids, filtering, and placing the filter cake at 20°C to 50°C to dry.

18. The preparation method according to claim 17, characterized in that: The solvent used in the preparation method is one or more mixed solvents of water, methanol, ethanol, isopropanol, n-propanol and acetone, and the amount of the solvent used is 1 to 100 times the weight ratio of levornidazole phosphate.

19. Use of the levoornidazole phosphate crystal form I according to claim 1 in the preparation of a medicament for treating infections caused by anaerobic bacteria.

Images